Enzymic microassay for measurement of chlorite, chlorate and perchlorate

ABSTRACT

An enzymatic assay for chlorite, chlorate and perchlorate determination based on horseradish peroxidase, chlorate reductase, or nitrate reductase and a reacting dye was developed. The assay is quick and simple requiring less than 30 min; is cheap costing less than $0.05 per sample; uses standard laboratory equipment; is sensitive to a lower limit of 0.4 micromolar and linear up to 6.0 millimolar; is highly reproducible and interference free. The application of this assay to chlorite analysis in treated water will significantly reduce one of the major costs associated with the use of chlorine dioxide as a disinfectant and improve the drinking water quality.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is related to, and claims a benefit of priorityunder 35 U.S.C. § 119(e) and/or 35 U.S.C. § 120 from, copending U.S.Serial. No. 60/386,894, filed Jun. 7, 2002, the entire contents of whichare hereby expressly incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

[0002] The government owns rights in the present invention pursuant togrant number DACA72-00-C-0016 from the US Department of Defense.

[0003] 1. Field of the Invention

[0004] The present invention relates generally to the fields analyticalchemistry and enzymology. More particularly, it concerns a new, rapidcalorimetric assay for the detection of chlorite in environmentalsamples.

[0005] 2. Description of Related Art

[0006] In the last two decades it has become apparent that the treatmentof drinking water with chlorine can result in the abiotic production ofpotentially toxic tribalomethanes (Rook, 1974; Bellar et al., 1974),some of which, such as chloroform, are known to be carcinogenic (NC1,1976). As a result, the use of chlorine dioxide for potable and wastewater treatment has been proposed and it is rapidly becoming thedisinfectant of choice amongst the drinking water providers throughoutthe U.S., Europe, Canada, and Japan. Current estimated annual productionof chlorine dioxide to satisfy this demand is approximately 1.7 millionmetric tonnes (D. J. Denby, Sterling Pulp Chemicals personalcommunication). Similarly to hypochlorite, chlorine dioxide is a strongdisinfectant over a wide pH range due to its strong oxidizing potential.It is effective in killing bacteria and deactivating viruses (Narkis etal., 1988; Narkis et al., 1989; Narkis and Kott, 1992). In addition, itis not known to form any tribalomethanes (Weinberg and Narkis, 1992) andhas been shown to significantly reduce odor and color in finished water(Gordon and Tachiyashiki, 1991).

[0007] However, the use of chlorine dioxide is carefully regulated dueto the toxicity of its active ingredient sodium chlorite (NaClO₂) and itmust be registered by various government organizations. In the UnitedStates, these may include EPA, FIFRA, FDA or USDA. Chlorite is regulatedunder stage 1 of the Disinfectant/Disinfection Byproduct Rule of the1996 Safe Drinking Water Act Amendments and continuous analysis forchlorite in treated water poses a prohibitive cost to smaller treatmentfacilities thus preventing their adoption of the superior disinfectantqualities of chlorine dioxide. This, and other guidelines are institutedbecause of the toxicity of chlorite to animals. Exposure to excessivedosages can severely damage organs and reduce the cellular and bloodlevel of glutathione which serves to protect cells from oxidizingagents. At concentrations at or above 100 mgL⁻¹ chlorite causes adecrease in red blood cell count, hemoglobin concentration and packedcell volume after a 30 day exposure (Couri et al., 1982). A MaximumContaminant Level (MCL) for chlorite has been established at 1 mgL⁻¹. Aspart of this, the EPA published a Direct Final Rule that approves theuse of updated versions of several ASTM, Standard Methods, andDepartment of Energy analytical methods for compliance monitoring ofcertain drinking water contaminants.

[0008] Currently the recognized method for reliable chloritedetermination is the use of ion chromatographic analysis withconductivity detection. However, this is an extremely costly andtime-consuming method that requires specialized-equipment and trainedpersonnel. As such, the adoption of chlorine dioxide as a disinfectantof choice by the Municipal Water treatment industry for drinking watersupplies is hesitant although it offers significant advantages overalternative disinfectants.

[0009] Therefore, it would be beneficial to provide a method forreliable chlorite determination that is quick, simple, and cheap.Preferably, this method should use standard laboratory equipment, have abroad linear working range for chlorite detection, be, reproducible,essentially interference free, and use a minimal amount of sample.

SUMMARY OF THE INVENTION

[0010] In accordance with the present invention, there is provided anassay for determining the presence of chlorite, chlorate and perchloratein a sample comprising: (a) providing a sample; (b) contacting saidsample with horseradish peroxidase (HRP) a calorimetric dye; (c)assessing color transformation, wherein color change indicates thepresence of chlorite in said sample mixture. The assay may be solidphase, using horseradish peroxidase fixed to a solid support, or it maybe conducted in solution. The volume of the sample mixture is about 10μl-5 ml. The time from step (b) to step (c) is about 5-45 min. Step (b)in the assay is performed at a temperature of about 20° C.-30° C. or insome examples the temperature is about 25° C. The assay also comprisesperforming a positive control reaction and a negative control reaction.

[0011] The assay method can be applied towards homogeneous orheterogeneous samples. Samples can also be solutions. The samples can befrom soil, water, sediment or wastestream. Where chlorate/perchlorate isto be measured, the assay is adapted by the addition of perchloratereductase or nitrate reductase.

[0012] The colorimetric dye used in the assay method can beo-dianisidine, lissamine green, methyl viologen, or similar dye whichchemically reacts with chlorine dioxide to give a measurable colorchange. The solid assays can be semi-quantitative. However, the liquidassay is highly quantitative. The detection range for the solid andliquid assay can be about 0.4 μm to 6000 μm. Sample mixture isapproximately at pH 7.0-7.5 for the assay. The solid phase assay supportcan be a column, a bead, or a dipstick which can be comprised of paper,plastic, diamatacious earth or alginate.

[0013] The present invention also provides kits for determining thepresence of chlorite, chlorate and perchlorate in a solid or liquidsample. The kits are comprised of horseradish peroxidase, or perchloratereductase or nitrate reductase, a calorimetric dye, and optionally acuvette or a solid support such as a dipstick, and/or additional agentsin a suitable container. The kits may also comprise suitably aliquotedstandards for chlorite determination and/or additional agentcompositions of the present invention, whether labeled or unlabeled, asmay be used to prepare a standard curve for a detection assay.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0015]FIG. 1: Dip-stick version of chlorite assay. Varyingconcentrations of chlorite from 0.001 mM to 5.0 mM can be seen.

[0016] FIGS. 2A-2B: FIG. 2A (increasing line) depicts the production ofchlorine dioxide after the addition of chlorite to HRP. FIG. 2B depictsthe production of a yellow product (ex. chlorine dioxide ando-dianisidine complex) in the same solution after HRP was inactivatedand o-dianisidine was added. FIG. 2A (decreasing line) also shows thedecreased absorbance at 395 nm indicating a decrease of chlorine dioxideafter addition of the dye.

[0017]FIG. 3: The absorbance at 450 nm is shown for variousconcentrations of sodium chlorite for solutions with pH of 4.5, 5.0,5.5, 6.0, 6.5, 7.0, and 7.5.

[0018]FIG. 4: The absorbance at 450 nm is shown for variousconcentrations of sodium chlorite for solutions with pH of 7.0, 7.1,7.2, 7.3, 7.4, and 7.5.

[0019] FIGS. 5A-5B: The absorbance at 450 nm is shown for variousconcentrations of sodium chlorite for solutions having an incubationtime of 5, 10, 15, 20, 30, 40, and 50 minutes. The temperature of theassay was 25° (FIG. 5A) and 37° C. (FIG. 5B).

[0020]FIG. 6: The reproducibility in absorbance measurements at 450 nmis shown for various concentrations of sodium chlorite by a triplicateassay of three separate weights.

[0021]FIG. 7: The absorbance at 450 nm is shown for 2 mM sodium chloritesolutions which also contain varying concentrations of sodium sulfate,sodium dithionite, sodium sulfide and sodium thiosulfate.

[0022]FIG. 8: The absorbance at 450 nm is shown for 2 mM sodium chloritesolution containing varying concentrations of sodium chlorate.

[0023]FIG. 9: The absorbance at 450 nm is shown for 2 mM sodium chloritesolution containing varying concentrations of nitrite and nitrate.

[0024]FIG. 10: The absorbance at 450 nm is shown for 2 mM sodiumchlorite solution containing varying concentrations of sodium chloride.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0025] 1. The Present Invention

[0026] A calorimetric assay for the determination of low levelconcentrations of chlorite, chlorate and perchlorate has been developedand is presented herein. The assay is based upon the enzymatic reactionbetween a reductase, horseradish peroxidase and chlorite, chlorate andperchlorate to form respectively chorine dioxide and chlorite. Thechlorine dioxide formed then reacts with a colorimetric dye such aso-dianisidine or lissamine green to form a colored complex. The ensuingcolor formed is directly proportional to the initial chloriteconcentration. In the case of the o-dianisidine a yellow product isproduced which can be determined spectrophotometrically at 450 nm. Thisassay has been optimized for pH, temperature and incubation time (pH 7.3for 20 min, at 25° C.). The assay is sensitive to chloriteconcentrations as low as 0.4 micromolar and is linear and reproducibleup to 6.0 millimolar. The assay does not suffer interference from commonions such as nitrate, nitrite, sulfate, ferric iron, bromate, or fromother oxyanions of chlorine namely perchlorate and chlorate. This assayis accurate, reproducible, and sensitive, and does not require the useof specialized equipment or extensive training. It can be applied in oneof two forms, either as a quantitative calorimetric assay or as asemi-quantitative dipstick.

[0027] This assay may be used for analyzing a wide variety of samples,such as a solution from soil, water, sediment or a wastestream. Thesample may be taken directly from the source, solubilized in a water orbuffer solution, or processed further before analysis. The volume ofsample used will be about 10 μl-5 ml.

[0028] The EPA has set a limit of 1 ppm for chlorite which is 15micromolar chlorite. This is detectable with the assay described herein.

[0029] 2. Chlorite Reduction

[0030] The assay of the current invention is based on the enzymaticreaction between chlorite and horseradish peroxidase (HRP). Datasuggests that the chlorite is converted to chlorine dioxide and chloridewhich is comparable to previously reported results. Enzymes other thanHRP that are capable of converting chlorite to chlorine dioxide may alsobe used in the current invention. Both chlorate and nitrate reductasesquantitatively convert chlorate/perchlorate to chlorite so they can notbe used for the measurement of chlorite.

[0031] The HRP or other enzyme may be purified before use or used asobtained. Generally, “purified” will refer to a specific enzyme or otherbiomolecule that has been subjected to fractionation to remove variousother proteins, polypeptides, or peptides, and which compositionsubstantially retains its activity, as may be assessed, for example, bya protein assays or as would be known to one of ordinary skill in theart for the desired enzyme.

[0032] The HRP may also be substantially purified. Where the term“substantially purified” is used, this will refer to a composition inwhich the specific protein, polypeptide, or peptide forms the majorcomponent of the composition, such as constituting about 50% of theproteins in the composition or more. In preferred embodiments, asubstantially purified protein will constitute more than 60%, 70%, 80%,90%, 95%, 99% or even more of the proteins in the composition. Theactivity of HRP may also be varied, depending on the source, (e.g. TypeI HRP has a lower activity and may be used). Concentrations of the HRPwill be varied accordingly.

[0033] Various methods for quantifying the degree of purification ofproteins, polypeptides, or peptides will be known to those of skill inthe art in light of the present disclosure. These include, for example,determining the specific protein activity of a fraction, or assessingthe number of polypeptides within a fraction by gel electrophoresis.

[0034] To purify a desired protein, polypeptide, or peptide a natural orrecombinant composition comprising at least some specific proteins,polypeptides, or peptides will be subjected to fractionation to removevarious other components from the composition. Various techniques aresuitable for use in protein purification will be well known to those ofskill in the art. These include, for example, precipitation withammonium sulfate, PEG, antibodies and the like or by heat denaturation,followed by centrifugation; chromatography steps such as ion exchange,ultrafiltration, dialysis, gel filtration, reverse phase,hydroxylapatite, lectin affinity and other affinity chromatographysteps; isoelectric focusing; gel electrophoresis; and combinations ofsuch and other techniques.

[0035] Although preferred for use in certain embodiments, there is nogeneral requirement that the protein, polypeptide, or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified protein, polypeptide or peptide, which arenonetheless enriched in the desired protein compositions, relative tothe natural state, will have utility in certain embodiments.

[0036] 3. Colorimetric Dyes

[0037] Conventional colorimetric assays are advantageous in that theyallow for direct, measurements of the color change. Absorbancemeasurements of color formation are simple, rapid, and can have a highrange of linearity.

[0038] Colorimetric dyes that may be used with HRP includeo-dianisidine, lissamine green, methyl viologen, 5-aminosalicylic acid(5AS), 2-2′ azino-di-(3-ethylbenzidine sulfonate (ABTS),o-phenylenediamine dihydrochloride (OPD), 3,3′,5-5′-tetramethylbenzidine 9TMB),N-(4-amino-5-methoxy-2-methylphenyl)benzamide (AMMB), orN-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) (Stewart et al., 2000).

[0039] Other dyes known in the art may be used if they chemically reactwith chlorine dioxide to give a measurable color change.

[0040] The wavelength for detection of the chlorite in the assay will bedetermined by the maximum absorbance of the calorimetric dye chosen, andcan be found by scanning a spectrometer over the applicable range andrecording the absorbance readings to determine the maxima.

[0041] 4. Solid Phase Assays

[0042] In one aspect of the invention, a solid phase assay will be usedin place of a cuvette and a solution phase assay. There are varioussupports for solid phase assays that can be used to determine thepresence of chlorite in a sample. The solid phase materials are knownfrom the art of separation science and chromatography. There are manykinds of chromatographic solid supports which may be used in the presentinvention. Examples of solid phase materials that may be used in thecurrent invention include a column, beads, or a dipstick. Variousmaterials may be used, e.g., paper, silica, hydroxyethyl cellulose,agarose, polyacrylamide, agarose-acrylamide, polyacrylamide, otherplastics, diamatacious earth, alginate and the like (Sambrook et al.,1989). The support may be modified for binding of horseradish peroxidaseor other enzyme useful in the current invention.

[0043] Generally, the specific support, pH, temperature and otherconditions are selected to maximize the chlorite detection and linearitywithin the particular conformation. The support may be a dipstick, aplate, a column, a microcolumn, a cuvette, or the like. The color changemay be detected by using a spectrophotometer or by visualization methodunder a suitable light source—the wavelength (i.e., UV, visible, or IR)is determined by the dye used.

[0044] 5. Kits

[0045] Any of the compositions described herein may be comprised in akit. In a non-limiting example, horseradish peroxidase, a calorimetricdye, and optionally a cuvette or a solid support such as a dipstick,and/or additional agent, may be comprised in a kit. The kits will thuscomprise, in suitable container means, horseradish peroxidase, acolorimetric dye, a support material and/or an additional agent of thepresent invention.

[0046] The kits may also comprise suitably aliquoted standards forchlorite determination and/or additional agent compositions of thepresent invention, whether labeled or unlabeled, as may be used toprepare a standard curve for a detection assay. The kit may comprisepositive and negative controls. The components of the kits may bepackaged either in aqueous media or other buffered solution. Thecontainer means of the kits will generally include at least onedipstick, column, plate, cuvette, vial, test tube, flask, bottle,syringe or other container means, into which a component may be placed,and preferably, suitably aliquoted. Where there are more than onecomponent in the kit, the kit also will generally contain a second,third or other additional container into which the additional componentsmay be separately placed. However, various combinations of componentsmay be comprised in a vial or cuvette. The kits of the present inventionalso will typically include a means for containing horseradishperoxidase, a calorimetric dye, an optional cuvette or support material,additional agent such as standards, positive controls and negativecontrols, and any other reagent containers in close confinement forcommercial sale. Such containers may include injection or blow-moldedplastic containers into which the desired vials are retained. The kitmay have a single container means, and/or it may have distinct containermeans for each compound.

[0047] When the components of the kit are provided in one and/or moreliquid solutions, the liquid solution is an aqueous solution, with asterile aqueous solution being particularly preferred. However, thecomponents of the kit may be provided as dried powder(s). When reagentsand/or components are provided as a dry powder, the powder can bereconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container means.

[0048] The container means will generally include at least one dipstick,column, plate, cuvette, vial, test tube, flask, bottle, syringe and/orother container means, into which the horseradish peroxidase or otherenzyme and the dye are placed, preferably, suitably allocated. The kitsmay also comprise a second container means for containing a sterile,pharmaceutically acceptable buffer and/or other diluent.

[0049] The kits of the present invention will also typically include ameans for containing the vials in close confinement for commercial sale,such as, e.g., injection and/or blow-molded plastic containers intowhich the desired vials are retained.

[0050] 6. Definitions

[0051] As used herein, the term sample refers to a solution containing aknown or unknown quantity of chlorite. The sample may be an aqueoussolution, a suspension, or other solution. The sample may be comprisedof soil, sediment or wastestream effluent.

[0052] As used herein, the term semi-quantitative refers to an assaythat is able to provide relative concentrations of one or moresubstituants in a sample. Semi-quantitative techniques can be used todetermine quantitative values by means of standards.

[0053] As used herein the specification, “a” or “an” may mean one ormore. As used herein in the claim(s), when used in conjunction with theword “comprising”, the words “a” or “an” may mean one or more than one.As used herein “another” may mean at least a second or more.

7. EXAMPLES

[0054] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Sample Solution Phase Chlorite Assay

[0055] Procedure for a 10 μL Assay. To prepare the HRP/dye mix, a 100 mMphosphate buffer with a pH 7.3 is made by combining 26.6 mL 1 MKH₂PO₄(monobasic), 73.4 mL 1M K₂HPO₄ (Dibasic) and is make up to 1 Lwith nanopure water and check the pH, it should be 7.3. This is storedin a cold room. Stock HRP (30 U/mL) is made by weighting out 3 mg HRP(stored in −20° C.) and gently mixing in 10 mL 100 mM phosphate buffer.This solution is gently mixed bu not shaken. The HRP/Dye mix is made bydiluting the stock to 1 U/mL in desired volume of 100 mM phosphatebuffer and adding o-dianisdine to a final concentration of 0.128 mM.This dye is stored in a cold room and typically one would add 1.6 mL dyeto 100 mL of dilute HRP. It is stable for 1 month if stored in the coldroom and stored in the dark.

[0056] A stock solution of sodium chlorite, usually 20 mM, in water(2.26 mg/mL) is prepared and dissolve up just before use. A standardcurve with stock chlorite in 0-5 mM range with final volume of 10 μL canbe prepared. 10 μL of sample to be tested or chlorite standard iscombined with 2 mL of HRP/Dye and left at room temperature for 20 min.The absorbance is read at 450 nm. If chlorite is present, there is ayellowish color.

[0057] Procedure for 2 mL Assay. For a larger volume assay, a 10 U/mLsolution of HRP in 100 mM phosphate buffer is prepared at pH 7.3.Chlorite standard or sample (1.8 mL) is added with 32 μL of 8 mMO-dianisdine. Then 200 μL of 10 U/mL HRP solution is added and left for20 min at room temperature. The absorbance is read at 450 nm.

Example 2 Solution-Phase Assay Set-Up

[0058] In one example, the assay is set up by taking 10 μL of a solutioncontaining chlorite and add 2 mL of HRP/Dye solution and incubate atroom temperature for 20 min. The optical density of the sample is readat 450 nm using a spectrophotometer. Unknown samples are determined froma standard curve prepared in the range of 0.02 nM to 5.0 nM chlorite.

[0059] In the first step, chlorite was added to HRP the production ofchlorine dioxide was followed spectrophotometrically at 395 nm. Thissolution was then heat treated to inactivate HRP. Next, on addition ofo-dianisidine to the heat treated solution, a yellow product developedwithin 20 secs (measured at 450 nm) and was accompanied by a concomitantdecrease in absorbance at 395 nm indicating that the dye reactsabiotically with the chlorine dioxide.

Example 3 Sample Solid-Phase Chlorite Assay

[0060] To a dip-stick containing horseradish peroxidase and acolorimetric dye along with an area for a water control sample varyingstandard concentrations of chlorite are added. The dip-stick is thendipped into a sample solution letting all areas of the dip-stick beexposed to the solution except the water control area. Next the dipstickis incubated at room temperature for 20 minutes. The dipstick can thenbe read visually or by use of a spectrophotometer to determine theconcentration of chlorite present in the sample solution.

Example 4 Optimization of Assay Conditions

[0061] The optimum pH for the chlorite assay was initially assessedusing a broad range of pH's as shown in FIG. 3. A 100 mM sodium acetatebuffer was used for pH 4.5 to 5.5 and a 100 mM potassium phosphatebuffer for pH's 6.0 to 7.5. The optimum pH range which exhibited thebest linearity was pH 7.0-7.5 (FIG. 4). The pH of the assay buffer (100mM phosphate) had a significant effect on color development. The optimumpH selected for the assay was pH 7.3 as it gave the most linear curveover the concentration range tested.

[0062] For other assay conditions, this optimization procedure may berepeated, and a different linear range may be found. For example, if atris buffer is used, the optimal pH range will most be between 7.5 and8.5.

[0063] The chlorite assay was optimized for time and temperature. Theassay was conducted at 37° C. (FIG. 5A) and 25° C. (FIG. 5B) and opticaldensity was read every 5 to 10 min. Analysis of FIGS. 5A-5B shows thatthere was no obvious difference between the two temperatures selected,therefore, the preferred temperature for the assay varies from about 20°C.-40° C. The optimum time for incubation was 20 min. After the 20 mintime, the linearity of the curve suffered although OD continued toincrease (FIGS. 5A-5B). Therefore, the reaction time for the chloratereduction is preferentially about 5-45 minutes, and is more preferablyabout 20 min for the current embodiment, however, shorter or longertimes may be appropriate depending on pH, sample concentrations, andother conditions.

[0064] In addition to the reaction time, a holding time betweencollection of the sample and spectrometric detection to allow fortransport of the sample to a lab or to collect a number of samplesbefore analysis. It is preferred that the sample be kept cold by ice orother means during this time. If chilled, the sample should be allowedto warm up to room temperature before spectrometric detection becausethe HRP reaction is temperature dependent. The holding time may be up to1, 2, 3, 4 or 5 hours.

[0065] It is also contemplated that a positive control reaction or anegative control reaction will be run with the assay reaction.

Example 5 Assay Characteristics

[0066] The chlorite assay is both linear and reproducible in theconcentration range of 0.02-6.0 mM as shown in FIG. 6. This test wasconducted with three separate weights of chlorite from which triplicateassays were performed.

Example 6 Effect of Contaminants

[0067] At a constant concentration of sodium chlorite (2 mM), the assaywas not affected by the presence of sulfate, but was inhibited byreducing agents such as dithionite, sulfide and thiosulfate (FIG. 7).This is not unexpected since sulfide is a known inhibitor of HRP and sopresumably is thiosulfate and dithionite.

[0068] Furthermore, at a constant concentration of sodium chlorite (2mM), the assay was not affected by the presence of other oxyanions ofchlorine such as perchlorate or chlorate over a wide concentration range(1.0-15.0 mM, FIG. 8).

[0069] At a constant concentration of sodium chlorite (2 mM), the assaywas not affected by the presence of nitrate or nitrate over a wideconcentration range (1.0-15.0 mM, FIG. 9).

[0070] At a constant concentration of sodium chlorite (2 mM), the assaywas not affected by the various concentrations of salt, as shown in FIG.10. This indicates that the assay is compatible for use with marinesamples.

Example 7 Applications

[0071] The chlorite assay has been successfully used by this lab duringthe purification of the enzyme chlorite dismutase from the (per)chloratereducing micro-organism Dechloromonas agitata str. CKB Since the assayonly requires a 10 μL sample, all fractions collected could be assayedfor activity. Thus, the assay allows for the identification andselection of which fractions contain the highest activity and thus thehighest concentration of active enzymes to purify. This resulted in a55-fold purification of the enzyme with a specific activity of 1928 μmolchlorite dismutated per min per mg protein.

Prophetic Example 8 Field use of Chlorite Assay

[0072] This assay can be adapted for use in the field using hand heldspectrophotometers. This will allow for rapid screening of areas forchlorite contamination and may be followed up with additional assays ina more sensitive laboratory setting if chlorite levels warrant furtherconcern. General techniques for adapting assays for field use can beused. These techniques may include the optimization for smallerspectrophotometers or spectrophotometers with lower sensitivity,producing a kit for the rapid screening at a test site where highlytrained clinicians are not required.

[0073] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and methods, and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

[0074] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0075] Bellar, Lichtenberg, Kroner, J. Am. Water Works Assoc.,66:703-706, 1974.

[0076] Couri, Abdel-Rahman, Bull, Environ. Health Perspect., 46:13-17,1982.

[0077] Gordon and Tachiyashiki, Environ. Sci, Technol., 25:468-474,1991.

[0078] Narkis and Kott, “Comparison between chlorine dioxide andchlorine for use as a disinfectant of wastewater effluents,”1-1483-1492, Pergamon, Wash. DC, 1992.

[0079] Narkis, Offer, Betzer, “The use of chlorine dioxide indisinfecting of advanced treated effluents,” Tel-Aviv, 1988.

[0080] Narkis, Offer, Linenberg, Betzer, In: WaterChlorination-Chemistry, Environmental Impact and Health Effects, Jolley(Eds.), Lewis Pub Inc., 955-966, Chelsa, Mich., 1989.

[0081] NCI, Report on carcinogenesis bioassay of chloroform (Natl.Cancer Institute), 1976.

[0082] Rook, J. Water Treat. Exam., 23:234-236, 1974.

[0083] Sambrook et al., “Molecular Cloning,” A Laboratory Manual, 2dEd., Cold Spring Harbor Laboratory Press, New York, 13.7-13.9:1989.

[0084] Stewart et al., Methods in Cell Science, 22:67-78, 2000.

[0085] Weinberg and Narkis, “Oxidation byproduct resulting from theinteraction of chlorine dioxide with nonionic surfactants,” VanderbiltUniv., TN, 1992.

What is claimed is:
 1. An assay for determining the presence of chlorite, chlorate and perchlorate in a sample comprising: (a) providing a sample; (b) contacting the sample with horseradish peroxidase (HRP) and a colorimetric dye; and (c) assessing color transformation, wherein color change indicates the presence of chlorite in the sample mixture.
 2. The assay of claim 1, wherein the assay is a solid phase assay.
 3. The assay of claim 2, wherein HRP is fixed to a solid support.
 4. The assay of claim 1, wherein the assay is conducted in solution.
 5. The assay of claim 1, wherein the volume of the sample mixture is about 10 μl-5 ml.
 6. The assay of claim 1, wherein the time from step (b) to step (c) is about 5-45 min.
 7. The assay of claim 1, wherein step (b) is performed at a temperature of about 20° C.-30° C.
 8. The assay of claim 7, wherein step (b) is performed at a temperature of about 25° C.
 9. The assay of claim 1, further comprising performing a positive control reaction.
 10. The assay of claim 1, further comprising performing a negative control reaction.
 11. The assay of claim 1, wherein the sample is homogeneous.
 12. The assay of claim 1, wherein the sample is heterogeneous.
 13. The assay of claim 1, wherein the sample is derived from soil, water, sediment or waste stream.
 14. The assay of claim 1, wherein the colorimetric dye is o-dianisidine, lissamine green, methyl viologen, or a similar dye which chemically reacts with chlorine dioxide to give a measurable color change.
 15. The assay of claim 2, wherein the solid assay is semi-quantitative.
 16. The assay of claim 4, wherein the liquid assay is quantitative.
 17. The assay of claim 1, wherein the detection range is about 0.4 μm to 6000 μm.
 18. The assay of claim, wherein the sample mixture is at a pH of approximately 7.0-7.5 for the assay.
 19. The assay of claim 3, wherein the solid phase support is a column, a bead, or a dipstick.
 20. The assay of claim 19, wherein the dipstick is comprised of paper, plastic, diamatacious earth or alginate.
 21. The assay of claim 1, wherein chlorate or perchlorate are measured, and step (b) further comprises contacting the sample with chlorate reductase or nitrate reductase. 